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AIMS: The mTOR/S6K1 signaling axis, known for cell growth regulation, is hyper-activated in multiple cancers. In this study, we have examined the mechanisms for ribosomal protein p70-S6 kinase 1 (S6K1) associated transformed human hepatocyte (THH) growth regulation. MAIN METHODS: THH were treated with p70-S6K1 inhibitor and analyzed for cell viability, cell cycle distribution, specific marker protein expression by western blot, and tumor inhibition in a xenograft mouse model. We validated our results by knockdown of p70-S6K1 using specific siRNA. KEY FINDINGS: p70-S6K1 inhibitor treatment caused impairment of in vitro hepatocyte growth, and arrested cell cycle progression at the G1 phase. Further, p70-S6K1 inhibitor treatment exhibited a decrease in FAK and Erk activation, followed by altered integrin-ß1 expression, caspase 8, and PARP cleavage appeared to be anoikis like growth inhibition. p70-S6K1 inhibitor also depolymerized actin microfilaments and diminished active Rac1/Cdc42 complex formation for loss of cellular attachment. Similar results were obtained with other transformed human hepatocyte cell lines. p70-S6K1 inhibition also resulted in a reduced phospho-EGFR, Slug and Twist; implicating an inhibition of epithelial-mesenchymal transition (EMT) state. A xenograft tumor model, generated from implanted THH in nude mice, following intraperitoneal injection of S6K1 inhibitor prevented further tumor growth. SIGNIFICANCE: Our results suggested that p70-S6K1 inhibition alters orchestration of cell cycle progression, induces cell detachment, and sensitizes hepatocyte growth impairment. Targeting p70 isoform of S6K1 by inhibitor may prove to be a promising approach together with other therapies for hepatocellular carcinoma (HCC) treatment.
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Anoikis , Hepatócitos/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Actinas/metabolismo , Animais , Western Blotting , Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/metabolismo , Ciclo Celular , Transição Epitelial-Mesenquimal , Imunofluorescência , Hepatócitos/fisiologia , Humanos , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas Experimentais/metabolismo , Camundongos Nus , Transplante de Neoplasias , Isoformas de Proteínas , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Fosfatases/fisiologia , Proteínas Quinases S6 Ribossômicas 70-kDa/antagonistas & inibidores , Proteínas Quinases S6 Ribossômicas 70-kDa/fisiologiaRESUMO
BACKGROUND: Cell surface protein, CD20, is extensively expressed on the surface of B cells. Antibodies targeting CD20 protein are being used to treat B-cell malignancies and B-cell mediated autoimmune diseases. Considering the cost of therapy with innovator monoclonal antibodies for these diseases, development of biosimilar products for the treatment of such diseases provides affordable solution to rising healthcare costs. MATERIALS AND METHODS: Reference products of rituximab (six batches) were procured and stored as per manufacturer's instructions. Cell lines used in bioassay were procured from American Type Culture Collection and all other reagents used for analysis were of analytical grade. Primary structure was studied by intact mass analysis, peptide fingerprinting, peptide mass fingerprinting and sequence coverage analysis. Higher order structure was studied by circular dichroism, ultraviolet-visible spectroscopy, fluorescence spectroscopy, and disulfide bridge analysis. Different isoforms of reference product and SB-02 were identified using capillary isoelectric focusing and capillary zone electrophoresis. Glycosylation was studied by N-glycan mapping using LC-ESI-MS, point of glycosylation, released glycan analysis using ultra performance liquid chromatography (UPLC). Product related impurities such as oligomer content analysis and oxidized impurities were studied using size exclusion chromatography and reverse phase high performance liquid chromatography, respectively. RESULTS AND CONCLUSION: Here, we report physicochemical and biological characterizations of Sun Pharma's proposed biosimilar (SB-02) to rituximab, a monoclonal anti-CD20 antibody approved for the treatment of non-Hodgkin's lymphoma and chronic lymphocytic leukemia. SB-02 and rituximab exhibited indistinguishable primary as well as higher-order structure upon analyzing with the array of analytical and extended characterization methods according to statistical methods. The molecule also displayed comparability to reference product in post-translational modifications and charge heterogeneity. In functional bioassays, SB-02 demonstrated comparable potency with respect to reference product. Our results indicate highly similar quality profile between SB-02 and rituximab.
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INTRODUCTION: Regulatory agencies recommend a stepwise approach for demonstrating biosimilarity between a proposed biosimilar and reference biological product emphasizing for functional and structural characterization to trace if there is any difference which may impact safety and efficacy. We studied the comparative structural and biological attributes of recombinant human chorionic gonadotropin (rhCG), SB005, with reference product, Ovidrel® and Ovitrelle®. Recombiant hCG was approved in 2000 by the US Food and Drug Administration for the induction of final follicular maturation, early luteinization in infertile women as part of assisted reproductive technology program. It is also indicated for the induction of ovulation and pregnancy in ovulatory infertile patients whose cause of infertility is not due to ovarian failure. MATERIALS AND METHODS: Primary structure was studied by intact mass analysis, peptide fingerprinting, peptide mass fingerprinting and sequence coverage analysis. Higher order structure was studied by circular dichroism, ultraviolet-visible spectroscopy, fluorescence spectroscopy, and disulfide bridge analysis. Different isoforms of reference product and SB005 were identified using capillary isoelectric focusing and capillary zone electrophoresis. Glycosylation was studied by N-glycan mapping using LC-ESI-MS, point of glycosylation, released glycan analysis using ultra performance liquid chromatography and sialic acid analysis. Product related impurities such as oligomer content analysis and oxidized impurities were studied using size exclusion chromatography and reverse phase high performance liquid chromatography, respectively. Biological activity in term of potency of reference product and SB005 was studied by in vivo analysis. RESULTS AND CONCLUSION: In this study we have compared analytical similarity of recombinant rhCG (SB005) produced at Sun Pharmaceuticals with the reference product with respect to its primary, higher order structure, isoforms, charge variants, glycosylation, sialyation pattern, pharmacodynamic and in vivo efficacy. Our studies show that the in house produced rhCG has a high degree of structural and functional similarity with the reference product available in the market.
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Exogenous glucocorticoid administration results in hyperglycemia, insulin resistance, hepatic dyslipidemia, and hypertension, a constellation of findings known as Cushing's syndrome. These effects are mediated by the glucocorticoid receptor (GR). Because GR activation in liver and adipose has been implicated in metabolic syndrome (MS), we wanted to determine the role of GR in these tissues in the development of MS. Because GR knockout (KO) mice (whole-body KO) exhibit perinatal lethality due to respiratory failure, we generated tissue-specific (liver or adipose) GRKO mice using cre-lox technology. Real-time PCR analysis of liver mRNA from dexamethasone-treated wildtype (WT) and liver GRKO mice indicated that hepatic GR regulates the expression of key genes involved in gluconeogenesis and glycogen metabolism. Interestingly, we have observed that liver-specific deletion of GR resulted in a significant increase in mRNA expression of key genes involved in gluconeogenesis and glycogen metabolism in kidney tissue, indicating a compensatory mechanism to maintain glucose homeostasis. We have also observed that GR plays an important role in regulating the mRNA expression of key genes involved in lipid metabolism. Liver GRKO mice demonstrated decreased fat mass and liver glycogen content compared with WT mice administered dexamethasone for 2 weeks. Adipose-specific deletion of GR did not alter glucose tolerance or insulin sensitivity of adipose GRKO mice compared with WT mice administrated dexamethasone. This indicates that liver GR might be more important in development of MS in dexamethasone-treated mice, whereas adipose GR plays a little role in these paradigms.
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Tecido Adiposo/metabolismo , Rim/metabolismo , Fígado/metabolismo , Síndrome Metabólica/metabolismo , Receptores de Glucocorticoides/metabolismo , Triglicerídeos/metabolismo , Animais , Composição Corporal/fisiologia , Gluconeogênese/genética , Resistência à Insulina/genética , Metabolismo dos Lipídeos/genética , Síndrome Metabólica/genética , Camundongos , Camundongos Knockout , Receptores de Glucocorticoides/genéticaRESUMO
UNLABELLED: We have previously reported that hepatitis C virus (HCV) infection of primary human hepatocytes (PHH) induces the epithelial mesenchymal transition (EMT) state and extends hepatocyte life span (S. K. Bose, K. Meyer, A. M. Di Bisceglie, R. B. Ray, and R. Ray, J Virol 86:13621-13628, 2012, http://dx.doi.org/10.1128/JVI.02016-12). These hepatocytes displayed sphere formation on ultralow binding plates and survived for more than 12 weeks. The sphere-forming hepatocytes expressed a number of cancer stem-like cell (CSC) markers, including high levels of the stem cell factor receptor c-Kit. The c-Kit receptor is regarded as one of the CSC markers in hepatocellular carcinoma (HCC). Analysis of c-Kit mRNA displayed a significant increase in the liver biopsy specimens of chronically HCV-infected patients. We also found c-Kit is highly expressed in transformed human hepatocytes (THH) infected in vitro with cell culture-grown HCV genotype 2a. Further studies suggested that HCV core protein significantly upregulates c-Kit expression at the transcriptional level. HCV infection of THH led to a significant increase in the number of spheres displayed on ultralow binding plates and in enhanced EMT and CSC markers and tumor growth in immunodeficient mice. The use of imatinib or dasatinib as a c-Kit inhibitor reduced the level of sphere-forming cells in culture. The sphere-forming cells were sensitive to treatment with sorafenib, a multikinase inhibitor, that is used for HCC treatment. Further, stattic, an inhibitor of the Stat3 molecule, induced sphere-forming cell death. A combination of sorafenib and stattic had a significantly stronger effect, leading to cell death. These results suggested that HCV infection potentiates CSC generation, and selected drugs can be targeted to efficiently inhibit cell growth. IMPORTANCE: HCV infection may develop into HCC as an end-stage liver disease. We focused on understanding the mechanism for the risk of HCC from chronic HCV infection and identified targets for treatment. HCV-infected primary and transformed human hepatocytes (PHH or THH) generated CSC. HCV-induced spheres were highly sensitive to cell death from sorafenib and stattic treatment. Thus, our study is highly significant for HCV-associated HCC, with the potential for developing a target-specific strategy for improved therapies.
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Carcinoma Hepatocelular/patologia , Hepacivirus/metabolismo , Hepatite C/patologia , Neoplasias Hepáticas/patologia , Células-Tronco Neoplásicas/citologia , Animais , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/virologia , Óxidos S-Cíclicos/farmacologia , Dasatinibe/antagonistas & inibidores , Transição Epitelial-Mesenquimal , Feminino , Hepatócitos/virologia , Humanos , Mesilato de Imatinib/antagonistas & inibidores , Neoplasias Hepáticas/virologia , Camundongos , Transplante de Neoplasias , Niacinamida/análogos & derivados , Niacinamida/farmacologia , Compostos de Fenilureia/farmacologia , Proteínas Proto-Oncogênicas c-kit/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-kit/genética , Proteínas Proto-Oncogênicas c-kit/metabolismo , RNA Mensageiro/análise , RNA Mensageiro/genética , Fator de Transcrição STAT3/antagonistas & inibidores , Sorafenibe , Esferoides Celulares/efeitos dos fármacos , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
Approximately 170 million people worldwide are chronically infected with hepatitis C virus (HCV). Chronic HCV infection is the leading cause for the development of liver fibrosis, cirrhosis, hepatocellular carcinoma (HCC) and is the primary cause for liver transplantation in the western world. Insulin resistance is one of the pathological features in patients with HCV infection and often leads to development of type II diabetes. Insulin resistance plays an important role in the development of various complications associated with HCV infection. Recent evidence indicates that HCV associated insulin resistance may result in hepatic fibrosis, steatosis, HCC and resistance to anti-viral treatment. Thus, HCV associated insulin resistance is a therapeutic target at any stage of HCV infection. HCV modulates normal cellular gene expression and interferes with the insulin signaling pathway. Various mechanisms have been proposed in regard to HCV mediated insulin resistance, involving up regulation of inflammatory cytokines, like tumor necrosis factor-α, phosphorylation of insulin-receptor substrate-1, Akt, up-regulation of gluconeogenic genes like glucose 6 phosphatase, phosphoenolpyruvate carboxykinase 2, and accumulation of lipid droplets. In this review, we summarize the available information on how HCV infection interferes with insulin signaling pathways resulting in insulin resistance.
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UNLABELLED: We have previously shown that hepatitis C virus (HCV) infection modulates the expression of forkhead box transcription factors, including FoxO1 and FoxA2, which play key roles in gluconeogenesis and ß-oxidation of fatty acid, respectively. The aim of the present study was to determine the role of forkhead box transcription factors in modulating lipid metabolism. HCV infection or core protein expression alone in transfected Huh7.5 cells increased expression of sterol regulatory element binding protein 1c (SREBP-1c) and its downstream target, fatty acid synthase (FASN), which are key proteins involved in lipid synthesis. Knockdown of FoxO1 by small interfering RNA in HCV-infected cells significantly decreased SREBP-1c and FASN expression. Further, HCV infection or core protein expression in Huh7.5 cells significantly decreased the expression of medium-chain acyl coenzyme A dehydrogenase (MCAD) and short-chain acyl coenzyme A dehydrogenase (SCAD), involved in the regulation of ß-oxidation of fatty acids. Ectopic expression of FoxA2 in HCV-infected cells rescued the expression of MCAD and SCAD. Oil red O and neutral lipid staining indicated that HCV infection significantly increases lipid accumulation compared to that in the mock-infected control. This was further verified by the increased expression of perilipin-2 and decreased activity of hormone-sensitive lipase (HSL) in HCV-infected hepatocytes, implying increased accumulation of neutral lipids. Knockdown of FoxO1 and ectopic expression of FoxA2 significantly decreased HCV replication. Taken together, these results suggest that HCV modulates forkhead box transcription factors which together increase lipid accumulation and promote viral replication. IMPORTANCE: Hepatic steatosis is a frequent complication associated with chronic HCV infection. Its presence is a key prognostic indicator associated with the progression to hepatic fibrosis and hepatocellular carcinoma. Several mechanisms have been proposed to account for the development of steatosis and fatty liver during HCV infection. We observed that HCV infection increases expression of both SREBP-1c and FASN. Further investigation suggested that the expression of SREBP-1c and FASN is controlled by the transcription factor FoxO1 during HCV infection. In addition, HCV infection significantly decreased both MCAD and SCAD expression, which is controlled by FoxA2. HCV infection also increased lipid droplet accumulation, increased perilipin-2 expression, and decreased HSL activity. Thus, knockdown of FoxO1 (decreased lipogenesis) and overexpression of FoxA2 (increased ß-oxidation) resulted in a significant disruption of the platform and, hence, a decrease in HCV genome replication. Thus, targeting of FoxO1 and FoxA2 might be useful in developing a therapeutic approach against HCV infection.
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Fatores de Transcrição Forkhead/genética , Hepacivirus/fisiologia , Hepatite C/metabolismo , Fator 3-beta Nuclear de Hepatócito/genética , Metabolismo dos Lipídeos , Acil-CoA Desidrogenase/genética , Acil-CoA Desidrogenase/metabolismo , Linhagem Celular , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Ácidos Graxos/metabolismo , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/metabolismo , Regulação da Expressão Gênica , Hepacivirus/genética , Hepatite C/enzimologia , Hepatite C/genética , Hepatite C/virologia , Fator 3-beta Nuclear de Hepatócito/metabolismo , Humanos , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Proteínas do Core Viral/genética , Proteínas do Core Viral/metabolismo , Replicação ViralRESUMO
UNLABELLED: Natural killer (NK) cells and the complement system play critical roles in the first line of defense against pathogens. The synthesis of complement components C4 and C3 is transcriptionally downregulated by hepatitis C virus (HCV) core and NS5A proteins, and this negative regulation is apparent in chronically HCV-infected patients. In this study, we have examined the potential contribution of an NK cell line as a model in regulating complement synthesis. Coculture of NK cells (NK3.3) with human hepatoma cells (Huh7.5) expressing HCV core or NS5A protein led to a significant increase in C4 and C3 complement synthesis via enhanced specific transcription factors. Reestablishment of complement protein expression was found to be mediated by direct interaction between NKG2D on NK cells and the hepatocyte protein major histocompatibility complex class I-related chains A and B (MICA/B) and not to be associated with specific cytokine signaling events. On the other hand, C4 and C3 synthesis remained impaired in a coculture of NK cells and Huh7.5 cells infected with cell culture-grown HCV. The association between these two cell types through NKG2D and MICA/B was examined further, with MICA/B expression in HCV-infected hepatocytes found to remain inhibited during coculture. Further experiments revealed that the HCV NS2 and NS5B proteins are responsible for the HCV-associated decrease in MICA/B. These results suggest that HCV disables a key receptor ligand in infected hepatoma cells, thereby inhibiting the ability of infected cells to respond to stimuli from NK cells to positively regulate complement synthesis. IMPORTANCE: The complement system contributes to the protection of the host from virus infection. However, the involvement of complement in viral hepatitis has not been well documented. Whether NK cells affect complement component expression in HCV-infected hepatocytes remains unknown. Here, we have shown how HCV subverts the ability of NK cells to positively mediate complement protein expression.
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Proteínas do Sistema Complemento/biossíntese , Proteínas do Sistema Complemento/imunologia , Hepacivirus/imunologia , Células Matadoras Naturais/imunologia , Linhagem Celular , Complemento C3/biossíntese , Complemento C3/genética , Complemento C4/biossíntese , Complemento C4/genética , Proteínas do Sistema Complemento/genética , Regulação da Expressão Gênica , Genótipo , Hepacivirus/genética , Hepacivirus/metabolismo , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/virologia , Ativação Linfocitária/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Ligação Proteica , Proteínas não Estruturais Virais/metabolismoRESUMO
CD55 limits excessive complement activation on the host cell surface by accelerating the decay of C3 convertases. In this study, we observed that hepatitis C virus (HCV) infection of hepatocytes or HCV core protein expression in transfected hepatocytes upregulated CD55 expression at the mRNA and protein levels. Further analysis suggested that the HCV core protein or full-length (FL) genome enhanced CD55 promoter activity in a luciferase-based assay, which was further augmented in the presence of interleukin-6. Mutation of the CREB or SP-1 binding site on the CD55 promoter impaired HCV core protein-mediated upregulation of CD55. HCV-infected or core protein-transfected Huh7.5 cells displayed greater viability in the presence of CD81 and CD55 antibodies and complement. Biochemical analysis revealed that CD55 was associated with cell culture-grown HCV after purification by sucrose density gradient ultracentrifugation. Consistent with this, a polyclonal antibody to CD55 captured cell culture-grown HCV. Blocking antibodies against CD55 or virus envelope glycoproteins in the presence of normal human serum as a source of complement inhibited HCV infection. The inhibition was enhanced in the presence of both the antibodies and serum complement. Collectively, these results suggest that HCV induces and associates with a negative regulator of the complement pathway, a likely mechanism for immune evasion.
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Antígenos CD55/metabolismo , Via Clássica do Complemento/imunologia , Hepatite C/imunologia , Hepatite C/metabolismo , Hepatócitos/metabolismo , Vírion/metabolismo , Análise de Variância , Anticorpos Antivirais/metabolismo , Sítios de Ligação/genética , Western Blotting , Antígenos CD55/genética , Linhagem Celular , Primers do DNA/genética , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Hepatócitos/virologia , Humanos , Interleucina-6/metabolismo , Luciferases , Reação em Cadeia da Polimerase em Tempo Real , UltracentrifugaçãoRESUMO
Hepatitis C virus (HCV)-mediated liver disease progression may reflect distinct molecular mechanisms for increased hepatocyte growth and hepatic stellate cell activation. In this study, we have observed that primary human hepatocytes, when infected in vitro with cell culture-grown HCV genotype 1a or 2a, display viral RNA and protein expression. Infected hepatocytes displayed a fibroblast-like shape and an extended life span. To understand the changes at the molecular level, we examined epithelial-mesenchymal transition (EMT) markers. Increased mRNA and protein expression levels of vimentin, snail, slug, and twist and a loss of the epithelial cell marker E-cadherin were observed. Snail and twist, when examined separately, were upregulated in chronically HCV-infected liver biopsy specimens, indicating an onset of an active EMT state in the infected liver. An increased expression level of fibroblast-specific protein 1 (FSP-1) in the infected hepatocytes was also evident, indicating a type 2 EMT state. Infected hepatocytes had significantly increased levels of phosphorylated ß-catenin (Ser(552)) as an EMT mediator, which translocated into the nucleus and activated Akt. The phosphorylation level of ß-catenin at Thr(41)/Ser(45) moieties was specifically higher in control than in HCV-infected hepatocytes, implicating an inactivation of ß-catenin. Together, these results suggested that primary human hepatocytes infected with cell culture-grown HCV display EMT via the activation of the Akt/ß-catenin signaling pathway. This observation may have implications for liver disease progression and therapeutic intervention strategies using inhibitory molecules.
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Transição Epitelial-Mesenquimal/fisiologia , Hepacivirus/fisiologia , Hepatócitos/citologia , Biomarcadores/metabolismo , Western Blotting , Linhagem Celular Transformada , Imunofluorescência , Hepatócitos/virologia , Humanos , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , beta Catenina/metabolismoRESUMO
Hepatocytes are the main source of hepatitis C virus (HCV) replication and contain the maximum viral load in an infected person. Chronic HCV infection is characterized by weak cellular immune responses to viral proteins. Cathepsin S is a lysosomal cysteine protease and controls HLA-DR-antigen complex presentation through the degradation of the invariant chain. In this study, we examined the effect of HCV proteins on cathepsin S expression and found it to be markedly decreased in dendritic cells (DCs) exposed to HCV or in hepatocytes expressing HCV proteins. The downregulation of cathepsin S was mediated by HCV core and NS5A proteins involving inhibition of the transcription factors interferon regulatory factor 1 (IRF-1) and upstream stimulatory factor 1 (USF-1) in gamma interferon (IFN-γ)-treated hepatocytes. Inhibition of cathepsin S by HCV proteins increased cell surface expression of the invariant chain. In addition, hepatocytes stably transfected with HCV core or NS5A inhibited HLA-DR expression. Together, these results suggested that HCV has an inhibitory role on cathepsin S-mediated major histocompatibility complex (MHC) class II maturation, which may contribute to weak immunogenicity of viral antigens in chronically infected humans.
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Antígenos de Diferenciação de Linfócitos B/metabolismo , Catepsinas/antagonistas & inibidores , Hepacivirus/patogenicidade , Hepatócitos/imunologia , Hepatócitos/virologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Apresentação de Antígeno , Catepsinas/genética , Diferenciação Celular , Linhagem Celular , Membrana Celular/imunologia , Células Dendríticas/imunologia , Células Dendríticas/patologia , Células Dendríticas/virologia , Regulação para Baixo , Antígenos HLA-DR/metabolismo , Hepacivirus/genética , Hepacivirus/imunologia , Hepatite C Crônica/imunologia , Hepatite C Crônica/patologia , Hepatite C Crônica/virologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Fator Regulador 1 de Interferon/genética , Fator Regulador 1 de Interferon/metabolismo , Transfecção , Fatores Estimuladores Upstream/genética , Fatores Estimuladores Upstream/metabolismo , Proteínas do Core Viral/genética , Proteínas do Core Viral/imunologia , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologiaRESUMO
Hepatitis C virus (HCV) infection significantly increases the prevalence of type 2 diabetes mellitus (T2DM). Insulin receptor substrate 1 (IRS-1) plays a key role in insulin signaling, thus enabling metabolic regulation in mammalian cells. We have previously shown that HCV infection modulates phosphorylation of Akt, a downstream target of IRS-1. In this study, we further examined the status of total IRS-1 and the downstream regulation of the Akt pathway in understanding mTOR/S6K1 signaling using HCV genotype 2a (clone JFH1)-infected hepatocytes. Inhibition of IRS-1 expression was observed in HCV-infected hepatocytes compared to that in a mock-infected control. The status of the tuberous sclerosis complex (TSC-1/TSC-2) was significantly decreased after HCV infection of human hepatocytes, showing a modulation of the downstream Akt pathway. Subsequent study indicated an increased level of Rheb and mTOR expression in HCV-infected hepatocytes. Interestingly, the phosphoS6K1 level was higher in HCV-infected hepatocytes, suggesting a novel mechanism for IRS-1 inhibition. Ectopic expression of TSC-1/TSC-2 significantly recovered the IRS-1 protein expression level in HCV-infected hepatocytes. Further analyses indicated that HCV core protein plays a significant role in modulating the mTOR/S6K1 signaling pathway. Proteasome inhibitor MG 132 recovered IRS-1 and TSC1/2 expression, suggesting that degradation occurred via the ubiquitin proteasome pathway. A functional consequence of IRS-1 inhibition was reflected in a decrease in GLUT4 protein expression and upregulation of the gluconeogenic enzyme PCK2 in HCV-infected hepatocytes. Together, these observations suggested that HCV infection activates the mTOR/S6K1 pathway in inhibiting IRS-1 function and perturbs glucose metabolism via downregulation of GLUT4 and upregulation of PCK2 for insulin resistance.
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Hepacivirus/patogenicidade , Proteínas Substratos do Receptor de Insulina/antagonistas & inibidores , Resistência à Insulina , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Glucose/metabolismo , Hepatócitos/metabolismo , Hepatócitos/virologia , HumanosRESUMO
The third component of human complement (C3) plays a central role in innate immune function as its activation is required to trigger classical as well as alternative complement pathways. In this study, we have observed that sera from patients chronically infected with hepatitis C virus (HCV) displayed significantly lower C3 levels than sera from healthy individuals. Liver biopsy specimens from the same patients also exhibited lower C3 mRNA expression than liver tissues from healthy donors. C3 mRNA level was reduced in hepatocytes upon infection with cell culture-grown HCV genotype 1a or 2a in vitro. Further analysis suggested that HCV core protein displayed a weak repression of C3 promoter activity by downregulating the transcription factor farnesoid X receptor (FXR). On the other hand, HCV NS5A protein strongly downregulated C3 promoter activity at the basal level or in the presence of interleukin-1ß (IL-1ß) as an inducer. In addition, the expression of the transcription factor CAAT/enhancer binding protein beta (C/EBP-ß), which binds to the IL-1/IL-6 response element in the C3 promoter, was inhibited in liver biopsy specimens. Furthermore, expression of C/EBP-ß was reduced in hepatocytes infected with cell culture-grown HCV, as well as in hepatocytes transfected with the NS5A genomic region of HCV. Together, these results underscore the role of HCV NS5A protein in impairing innate immune function.
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Complemento C3/genética , Regulação para Baixo , Hepacivirus/metabolismo , Hepatite C/genética , Proteínas do Core Viral/metabolismo , Proteínas não Estruturais Virais/metabolismo , Animais , Linhagem Celular Tumoral , Complemento C3/imunologia , Regulação da Expressão Gênica , Hepacivirus/classificação , Hepacivirus/genética , Hepatite C/imunologia , Hepatite C/virologia , Humanos , Masculino , Regiões Promotoras Genéticas , Proteínas do Core Viral/genética , Proteínas não Estruturais Virais/genéticaRESUMO
Fifty-three cases of ventral hernia admitted between 15th July, 1993 and 31st December, 2001 in the IPGME&R, Kolkata is being presented with analysis of causes, method of repair and complications. The study was intended to find a simple, universal cost-effective repair of the deformity. Most cases can be repaired with a method of closure consisting of plication of the inner layer and 2 to 3 layered darning in the outer layer with '0' prolene. The main factor appears to be a tensionless closure and avoidance of postoperative infection and stress like cough and constipation. While in the west, use of prolene mesh has become a routine, the authors feel that darning is not only less expensive but reduces the amount of foreign body introduced.